2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol and its uses

ABSTRACT

This invention discloses 2α-methyl-19-nor-(20S)-vitamin D analogs, and specifically 2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol and pharmaceutical uses therefor. This compound exhibits pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent and for the treatment of skin diseases such as psoriasis as well as skin conditions such as wrinkles, slack skin, dry skin and insufficient sebum secretion. This compound also has little, if any, calcemic activity and therefore may be used to treat autoimmune disorders or inflammatory diseases in humans as well as renal osteodystrophy. This compound may also be used for the treatment or prevention of obesity.

This application is a continuation of U.S. patent application Ser. No.11/283,163, filed Nov. 18, 2005, now U.S. Pat. No. 7,241,751.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/630,184, filed Nov. 22, 2004.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol and itspharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferolis described in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

SUMMARY OF THE INVENTION

The present invention is directed toward 2α-methyl-19-nor-(20S)-vitaminD analogs, and more specifically toward2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol, theirbiological activity, and various pharmaceutical uses for thesecompounds. These new 1α-hydroxylated vitamin D compounds not knownheretofore are the 19-nor-vitamin D analogs having a methyl group at the2-position, and sec-butyl substituent at the 17-position (C-17).

Structurally these 2α-methyl-19-nor-(20S)-vitamin D analogs arecharacterized by the general formula I shown below:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group. The preferred analog is2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol which has thefollowing formula Ia:

The above compounds I, particularly Ia, exhibit a desired, and highlyadvantageous, pattern of biological activity. These compounds arecharacterized by relatively high binding to vitamin D receptors, butvery low intestinal calcium transport activity, as compared to that of1α,25-dihydroxyvitamin D₃, and have very low ability to mobilize calciumfrom bone, as compared to 1α,25-dihydroxyvitamin D₃. Hence, thesecompounds can be characterized as having little, if any, calcemicactivity. It is undesirable to raise serum calcium to supraphysiologiclevels when suppressing the preproparathyroid hormone gene (Darwish &DeLuca, Arch. Biochem. Biophys. 365, 123-130, 1999) and parathyroidgland proliferation. These analogs having little or no calcemic activitywhile very active on differentiation are expected to be useful as atherapy for suppression of secondary hyperparathyroidism of renalosteodystrophy.

The compounds I, particularly Ia, of the invention have also beendiscovered to be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compounds of the invention.

The above compounds I, and particularly Ia, are also characterized byrelatively high cell differentiation activity. Thus, these compoundsalso provide a therapeutic agent for the treatment of psoriasis, or asan anti-cancer agent, especially against leukemia, colon cancer, breastcancer, skin cancer and prostate cancer. In addition, due to theirrelatively high cell differentiation activity, these compounds provide atherapeutic agent for the treatment of various skin conditions includingwrinkles, lack of adequate dermal hydration, i.e. dry skin, lack ofadequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of these compounds thus not only results in moisturizingof skin but also improves the barrier function of skin.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of one or more of the compounds or thepharmaceutical compositions to the subject inhibits adipocytedifferentiation, inhibits gene transcription, and/or reduces body fat inthe animal subject.

One or more of the compounds may be present in a composition to treatthe above-noted diseases and disorders in an amount from about 0.01μg/gm to about 1000 μg/gm of the composition, preferably from about 0.1μg/gm to about 500 μg/gm of the composition, and may be administeredtopically, transdermally, orally, rectally, nasally, sublingually orparenterally in dosages of from about 0.01 μg/day to about 1000 μg/day,preferably from about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 illustrate various biological activities of2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol, hereinafterreferred to as “2α-methylbisP,” as compared to the native hormoneα,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of 2α-methylbisPand 1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of 2α-methylbisP and 1,25(OH)₂D₃;

FIG. 3 is a graph illustrating the in vitro transcription activity of1,25(OH)₂D₃ as compared to 2α-methylbisP;

FIG. 4 is a bar graph illustrating the bone calcium mobilizationactivity of 1,25(OH)₂D₃ as compared to 2α-methylbisP; and

FIG. 5 is a bar graph illustrating the intestinal calcium transportactivity of 1,25(OH)₂D₃ as compared to 2α-methylbisP.

DETAILED DESCRIPTION OF THE INVENTION

2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol (referred toherein as 2α-methylbisP) a 19-nor vitamin D analog which ischaracterized by the presence of a methyl substituent at the carbon 2(C-2), and sec-butyl substituent at the carbon 17 (C-17), wassynthesized and tested. Such vitamin D analog seemed an interestingtarget because the relatively small methyl group at the C-2 positionshould not interfere with binding to the vitamin D receptor.Structurally, this 19-nor analog is characterized by the general formulaIa previously illustrated herein, and its pro-drug (in protected hydroxyform) is characterized by general formula I previously illustratedherein.

The preparation of2α-methyl-19-nor-(20S)-1α-hydroxy-bishomopregnacalciferol analogs havingthe structure I can be accomplished by a common general method, i.e. theselective homogeneous catalytic hydrogenation of the exomethylene unitat carbon 2 in the 2-methylene-19-nor-1α-hydroxy-bishomopregnacalciferolcompounds of the general formula II performed efficiently in thepresence of tris(triphenylphosphine)rhodium(I) chloride [Wilkinson'scatalyst, (Ph₃P)₃RhCl]. Such reduction conditions allowed to reduce onlyC(2)=CH₂ unit leaving C(5)-C(8) butadiene moiety unaffected. Theisolated material is an epimeric mixture (ca. 1:1) of2-methyl-19-nor-vitamins I and III differing in configuration at C-2.The mixture can be used without separation or, if desired, theindividual 2α-(formula I) and 2β-(formula III) isomers can be separatedby an efficient HPLC system.

In the structures I, II and III, substituents X₁ and X₂ represent thegroups defined above.

2-methylene-19-nor-bishomopregnacalciferol analogs of the generalstructure II are known, or can be prepared by known methods.

The overall process of the synthesis of compounds I, II and III isillustrated and described more completely in U.S. Pat. No. 5,843,928entitled “2-Alkylidene-19-Nor-Vitamin D Compounds” the specification ofwhich is specifically incorporated herein by reference.

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively.

More specifically, reference should be made to the followingillustrative example and description as well as to Scheme 1 herein for adetailed illustration of the preparation of compound 2α-methylbisP.

In this example specific products identified by Arabic numerals (1, 2,3) refer to the specific structures so identified in the Scheme I.

EXAMPLE

Chemistry. Ultraviolet (UV) absorption spectra were recorded with aHitachi Model 60-100 UV-vis spectrometer in the solvent noted. ¹Hnuclear magnetic resonance (NMR) spectra were recorded at 500 MHz with aBruker AM-500 FT spectrometer in deuteriochloroform. Chemical shifts (δ)are reported downfield from internal Me₄Si (δ0.00). Mass spectra wererecorded at 70 eV on a Kratos DS-50 TC instrument equipped with a KratosMS-55 data system. Samples were introduced into the ion sourcemaintained at 120-250° C. via a direct insertion probe. High-performanceliquid chromatography (HPLC) was performed on a Waters Associates liquidchromatograph equipped with a Model 6000A solvent delivery system, aModel 6 UK Universal injector, a Model 486 tunable absorbance detector,and a differential R 401 refractometer.

Example 1 Hydrogenation of2-methylene-19-nor-(20S)-1α-hydroxy-bis-homo-pregnacalciferol (1)

Tris(triphenylphosphine)rhodium (I) chloride (29.0 mg, 31.3 μmol) wasadded to dry benzene (30 mL) presaturated with hydrogen (for 20 min).The mixture was stirred at room temperature until a homogeneous solutionwas formed (ca. 50 min). A solution of vitamin 1 (10 mg, 29.0 μmol) indry benzene (4 mL) was then added and the reaction was allowed toproceed under a continuous stream of hydrogen for 3.5 h. Benzene wasremoved under vacuum, the residue was redissolved in hexane/ethylacetate (7:3) and applied on Waters silica Sep-Pak (Vac 20 cc). Lesspolar impurities were eluted with the same solvent system (30 mL), and amixture of 2-methyl vitamins was eluted with hexane/ethyl acetate(65:35, 10 mL) and hexane/ethyl acetate (6:4, 20 mL). The combinedfractions were evaporated to give crude products (ca. 11 mg) which werefurther purified by HPLC (10 mm×25 cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (90:10) solvent system. The mixture (ca. 1:1) of both2α- and 2β-methyl-19-norvitamins 2 and 3 (6.85 mg, 69%) gave a singlepeak at R_(V) 28 mL (the starting 2-methylene compound 1 was eluted atR_(V) 26 mL in the same system). Separation of both epimers was achievedby reversed-phase HPLC (6.2 mm×25 cm Zorbax-ODS column, 2 mL/min) usingmethanol/water (90:10) solvent system. 2α-Methyl vitamin 3 (2.99 mg,30%) was collected at R_(V) 24 mL and its 2α-epimer 2 (3.46 mg, 34%) atR_(V) 28 mL (the starting 2-methylene compound 1 was eluted at R_(V) 27mL in the same system).

2: U (in EtOH) λ_(max) 242.0, 250.0, 260.0 nm; ¹H NMR (CDCl₃) δ 0.531(3H, s, 18-H₃), 0.827 (3H, d, J˜5.5 Hz, 21-H₃), 0.834 (3H, t, J=7.2 Hz,23-H₃), 1.134 (3H, d, J=6.9 Hz, 2α-CH₃), 2.13 (1H, ˜t, J˜11 Hz, 4β-H),2.22 (1H, br d, J˜13 Hz, 10β-H), 2.60 (1H, dd, J=12.7, 4.2 Hz, 4α-H),2.80 (2H, m, 9β- and 10α-H), 3.61 (1H, m, w/2=25 Hz, 3α-H), 3.96 (1H, m,w/2=12 Hz, 1β-H), 5.82 and 6.37 (1H and 1H, each d, J=11.2 Hz, 7- and6-H); MS m/z (relative intensity) 346 (M⁺, 100), 317 (16), 289 (39), 253(18), 229 (35), 191 (56), 135 (59), 91 (64); exact mass calcd forC₂₃H₃₈O₂ 346.2872. found 346.2857.

The introduction of a methyl group to the 2-position, and theelimination of carbons 24, 25, 26 and 27 in the side chain of1α-hydroxy-19-nor-vitamin D₃ had little binding to the full lengthrecombinant rat vitamin D receptor, as compared to1α,25-dihydroxyvitamin D₃. The compound 2α-methylbisP bound slightlyless to the receptor as compared to the standard 1,25-(OH)₂D₃ (FIG. 1).It might be expected from these results that compound 2α-methylbisPwould have equivalent biological activity. Surprisingly, however,compound 2α-methylbisP is a highly selective analog with uniquebiological activity.

FIG. 5 shows that 2α-methylbisP has very little activity as compared tothat of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural hormone, instimulating intestinal calcium transport.

FIG. 4 demonstrates that 2α-methylbisP has very little bone calciummobilization activity, as compared to 1,25(OH)₂D₃.

FIGS. 4 and 5 thus illustrate that 2α-methylbisP may be characterized ashaving little, if any, calcemic activity.

FIG. 2 illustrates that 2α-methylbisP is almost as potent as 1,25(OH)₂D₃on HL-60 cell differentiation, making it an excellent candidate for thetreatment of psoriasis and cancer, especially against leukemia, coloncancer, breast cancer, skin cancer and prostate cancer. In addition, dueto its relatively high cell differentiation activity, this compoundprovides a therapeutic agent for the treatment of various skinconditions including wrinkles, lack of adequate dermal hydration, i.e.dry skin, lack of adequate skin firmness, i.e. slack skin, andinsufficient sebum secretion. Use of this compound thus not only resultsin moisturizing of skin but also improves the barrier function of skin.

FIG. 3 illustrates that the compound 2α-methylbisP has about the sametranscriptional activity as 1α,25-dihydroxyvitamin D₃ in bone cells.This result, together with the cell differentiation activity of FIG. 2,suggests that 2α-methylbisP will be very effective in psoriasis becauseit has direct cellular activity in causing cell differentiation, genetranscription, and in suppressing cell growth. These data also indicatethat 2α-methylbisP may have significant activity as an anti-canceragent, especially against leukemia, colon cancer, breast cancer, skincancer and prostate cancer.

The strong activity of 2α-methylbisP on HL-60 differentiation suggestsit will be active in suppressing growth of parathyroid glands and in thesuppression of the preproparathyroid gene.

Experimental Methods

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wereoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined

using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient=42,000 and λ_(max)=252 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In Vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24 Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.

RLU=relative luciferase units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (0.47% Ca)diet+AEK for one week followed by Diet 11 (0.02% Ca)+AEK for 3 weeks.The rats were then switched to a diet containing 0.47% Ca for one weekfollowed by two weeks on a diet containing 0.02% Ca. Dose administrationbegan during the last week on 0.02% calcium diet. Four consecutive ipdoses were given approximately 24 hours apart. Twenty-four hours afterthe last dose, blood was collected from the severed neck and theconcentration of serum calcium determined as a measure of bone calciummobilization. The first 10 cm of the intestine was also collected forintestinal calcium transport analysis using the everted gut sac method.

Interpretation of Data

VDR binding, HL60 cell differentiation, and transcription activity.2α-methylbisP (K_(i)=1.6×10⁻⁹M) is slightly less active than the naturalhormone 1α,25-dihydroxyvitamin D₃ (K_(i)=1.8×10⁻¹⁰M) in its ability tocompete with [³H]-1,25(OH)₂D₃ for binding to the full-length recombinantrat vitamin D receptor (FIG. 1). There is also little difference between2α-methylbisP (EC₅₀=3.7×10⁻⁸M) in its ability (efficacy or potency) topromote HL60 differentiation as compared to 1α,25-dihydroxyvitamin D₃(EC₅₀=3.6×10⁻⁹M) (See FIG. 2). Also, compound 2α-methylbisP(EC₅₀=4.4×10⁻⁹M) has similar transcriptional activity in bone cells as1α,25-dihydroxyvitamin D₃ (EC⁵⁰⁻=2.9×10⁻⁹M) (See FIG. 3). These resultssuggest that 2α-methylbisP will be very effective in psoriasis becauseit has direct cellular activity in causing cell differentiation, genetranscription, and in suppressing cell growth. These data also indicatethat 2α-methylbisP will have significant activity as an anti-canceragent, especially against leukemia, colon cancer, breast cancer, skincancer and prostate cancer, as well as against skin conditions such asdry skin (lack of dermal hydration), undue skin slackness (insufficientskin firmness), insufficient sebum secretion and wrinkles. It would alsobe expected to be very active in suppressing secondaryhyperparathyroidism.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of 2α-methylbisP and 1,25(OH)₂D₃ inintestine and bone were tested. As expected, the native hormone(1,25(OH)₂D₃) increased serum calcium levels at all dosages (FIG. 4).FIG. 4 shows that 2α-methylbisP has little, if any, activity inmobilizing calcium from bone. Administration of 2α-methylbisP at 87pmol/day for 4 consecutive days did not result in mobilization of bonecalcium, and increasing the amount of 2α-methylbisP to 260 pmol/day andthen to 780 pmol/day and finally to 7020 pmol/day was also without anysubstantial effect.

Intestinal calcium transport was evaluated in the same groups of animalsusing the everted gut sac method (FIG. 5). These results show that thecompound 2α-methylbisP does not promote intestinal calcium transportwhen administered at 87 pmol/day, 260 pmol/day or 780 pmol/day, whereas1,25(OH)₂D₃ promotes a significant increase at the 260 pmol/day dose. Itwas only when 2340 pmol/day of 2α-methylbisP was administered thatsignificant intestinal calcium transport activity was recorded, analmost 10-fold increase in dosage over the 260 pmol/day dose. Thus, itmay be concluded that 2α-methylbisP is essentially devoid of intestinalcalcium transport activity at the recommended lower doses.

These results illustrate that 2α-methylbisP is an excellent candidatefor numerous human therapies as described herein, and that it may beparticularly useful in a number of circumstances such as suppression ofsecondary hyperparathyroidism of renal osteodystrophy, autoimmunediseases, cancer, and psoriasis. 2α-methylbisP is an excellent candidatefor treating psoriasis because: (1) it has significant VDR binding,transcription activity and cellular differentiation activity; (2) it isdevoid of hypercalcemic liability unlike 1,25(OH)₂D₃; and (3) it iseasily synthesized. Since 2α-methylbisP has significant binding activityto the vitamin D receptor, but has little ability to raise blood serumcalcium, it may also be particularly useful for the treatment ofsecondary hyperparathyroidism of renal osteodystrophy.

These data also indicate that the compound 2α-methylbisP of theinvention may be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compound 2α-methylbisP of the invention.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of the compound or the pharmaceuticalcompositions to the subject inhibits adipocyte differentiation, inhibitsgene transcription, and/or reduces body fat in the animal subject. Theanimal may be a human, a domestic animal such as a dog or a cat, or anagricultural animal, especially those that provide meat for humanconsumption, such as fowl like chickens, turkeys, pheasant or quail, aswell as bovine, ovine, caprine, or porcine animals.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I may be formulated for pharmaceuticalapplications as a solution in innocuous solvents, or as an emulsion,suspension or dispersion in suitable solvents or carriers, or as pills,tablets or capsules, together with solid carriers, according toconventional methods known in the art. Any such formulations may alsocontain other pharmaceutically-acceptable and non-toxic excipients suchas stabilizers, anti-oxidants, binders, coloring agents or emulsifyingor taste-modifying agents.

The compounds of formula I and particularly 2α-methylbisP, may beadministered orally, topically, parenterally, rectally, nasally,sublingually or transdermally. The compound is advantageouslyadministered by injection or by intravenous infusion or suitable sterilesolutions, or in the form of liquid or solid doses via the alimentarycanal, or in the form of creams, ointments, patches, or similar vehiclessuitable for transdermal applications. A dose of from 0.01 μg to 1000 μgper day of the compounds I, particularly 2α-methylbisP, preferably fromabout 0.1 μg to about 500 μg per day, is appropriate for preventionand/or treatment purposes, such dose being adjusted according to thedisease to be treated, its severity and the response of the subject asis well understood in the art. Since the compound exhibits specificityof action, each may be suitably administered alone, or together withgraded doses of another active vitamin D compound—e.g. 1α-hydroxyvitaminD₂ or D₃, or 1α,25-dihydroxyvitamin D₃—in situations where differentdegrees of bone mineral mobilization and calcium transport stimulationis found to be advantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds I, particularly 2α-methylbisP, asdefined by the above formula I and Ia as the active ingredient, and asuitable carrier. An effective amount of such compound for use inaccordance with this invention is from about 0.01 μg to about 1000 μgper gm of composition, preferably from about 0.1 μg to about 500 μg pergram of composition, and may be administered topically, transdermally,orally or parenterally in dosages of from about 0.01 μg/day to about1000 μg/day, and preferably from about 0.1 μg/day to about 500 μg/day.

The compounds I, particularly 2α-methylbisP, may be formulated ascreams, lotions, ointments, topical patches, pills, capsules or tablets,suppositories, aerosols, or in liquid form as solutions, emulsions,dispersions, or suspensions in pharmaceutically innocuous and acceptablesolvent or oils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, particularly 2α-methylbisP, may be advantageouslyadministered in amounts sufficient to effect the differentiation ofpromyelocytes to normal macrophages. Dosages as described above aresuitable, it being understood that the amounts given are to be adjustedin accordance with the severity of the disease, and the condition andresponse of the subject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group, wherein saidhydroxy-protecting group is an alkoxycarbonyl group, an acyl group, analkylsilyl group, an alkylarylsilyl group, or an alkoxyakyl group. 2.The compound of claim 1 wherein X₂ is hydrogen.
 3. The compound of claim1 wherein X₁ is hydrogen.
 4. The compound of claim 1 wherein X₁ and X₂are both t-butyldimethylsilyl.
 5. A pharmaceutical compositioncontaining an effective amount of at least one compound as claimed inclaim 1 together with a pharmaceutically acceptable excipient.
 6. Thepharmaceutical composition of claim 5 wherein said effective amountcomprises from about 0.01 μg to about 1000 μg per gram of composition.7. The pharmaceutical composition of claim 5 wherein said effectiveamount comprises from about 0.1 μg to about 500 μg per gram ofcomposition.
 8. A method of treating psoriasis comprising administeringto a subject with psoriasis an effective amount of a compound having theformula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group, wherein saidhydroxy-protecting group is an alkoxycarbonyl group, an acyl group, analkylsilyl group, an alkylarylsilyl group, or an alkoxyakyl group. 9.The method of claim 8 wherein the compound is administered orally. 10.The method of claim 8 wherein the compound is administered parenterally.11. The method of claim 8 wherein the compound is administeredtransdermally.
 12. The method of claim 8 wherein the compound isadministered topically.
 13. The method of claim 8 wherein the compoundis administered rectally.
 14. The method of claim 8 wherein the compoundis administered nasally.
 15. The method of claim 8 wherein the compoundis administered sublingually.
 16. The method of claim 8 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 17. A method of treating a disease selected from the groupconsisting of leukemia, colon cancer, breast cancer, skin cancer orprostate cancer comprising administering to a subject with said diseasean effective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group, wherein saidhydroxy-protecting group is an alkoxycarbonyl group, an acyl group, analkylsilyl group, an alkylarylsilyl group, or an alkoxyakyl group. 18.The method of claim 17 wherein the compound is administered orally. 19.The method of claim 17 wherein the compound is administeredparenterally.
 20. The method of claim 17 wherein the compound isadministered transdermally.
 21. The method of claim 17 wherein thecompound is administered rectally.
 22. The method of claim 17 whereinthe compound is administered nasally.
 23. The method of claim 17 whereinthe compound is administered sublingually.
 24. The method of claim 17wherein the compound is administered in a dosage of from about 0.01μg/day to about 1000 μg/day.
 25. A method of treating an autoimmunedisease selected from the group consisting of multiple sclerosis, lupus,diabetes mellitus, host versus graft rejection, and rejection of organtransplants, comprising administering to a subject with said disease aneffective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group, wherein saidhydroxy-protecting group is an alkoxycarbonyl group, an acyl group, analkylsilyl group, an alkylarylsilyl group, or an alkoxyakyl group. 26.The method of claim 25 wherein the compound is administered orally. 27.The method of claim 25 wherein the compound is administeredparenterally.
 28. The method of claim 25 wherein the compound isadministered transdermally.
 29. The method of claim 25 wherein thecompound is administered rectally.
 30. The method of claim 25 whereinthe compound is administered nasally.
 31. The method of claim 25 whereinthe compound is administered sublingually.
 32. The method of claim 25wherein the compound is administered in a dosage of from about 0.01μg/day to about 1000 μg/day.
 33. A method of treating an inflammatorydisease selected from the group consisting of rheumatoid arthritis,asthma, and inflammatory bowel diseases, comprising administering to asubject with said disease an effective amount of a compound having theformula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group, wherein saidhydroxy-protecting group is an alkoxycarbonyl group, an acyl group, analkylsilyl group, an alkylarylsilyl group, or an alkoxyakyl group. 34.The method of claim 33 wherein the compound is administered orally. 35.The method of claim 33 wherein the compound is administeredparenterally.
 36. The method of claim 33 wherein the compound isadministered transdermally.
 37. The method of claim 33 wherein thecompound is administered rectally.
 38. The method of claim 33 whereinthe compound is administered nasally.
 39. The method of claim 33 whereinthe compound is administered sublingually.
 40. The method of claim 33wherein the compound is administered in a dosage of from about 0.01μg/day to about 1000 μg/day.
 41. A method of treating a skin conditionselected from the group consisting of wrinkles, lack of adequate skinfirmness, lack of adequate dermal hydration and insufficient sebumsecretion which comprises administering to a subject with said skincondition an effective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group, wherein saidhydroxy-protecting group is an alkoxycarbonyl group, an acyl group, analkylsilyl group, an alkylarylsilyl group, or an alkoxyakyl group. 42.The method of claim 41 wherein the compound is administered orally. 43.The method of claim 41 wherein the compound is administeredparenterally.
 44. The method of claim 41 wherein the compound isadministered transdermally.
 45. The method of claim 41 wherein thecompound is administered topically.
 46. The method of claim 41 whereinthe compound is administered rectally.
 47. The method of claim 41wherein the compound is administered nasally.
 48. The method of claim 41wherein the compound is administered sublingually.
 49. The method ofclaim 41 wherein the compound is administered in a dosage of from about0.01 μg/day to about 1000 μg/day.
 50. A method of treating renalosteodystrophy comprising administering to a subject with renalosteodystrophy an effective amount of a compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group, wherein saidhydroxy-protecting group is an alkoxycarbonyl group, an acyl group, analkylsilyl group, an alkylarylsilyl group, or an alkoxyakyl group. 51.The method of claim 50 wherein the compound is administered orally. 52.The method of claim 50 wherein the compound is administeredparenterally.
 53. The method of claim 50 wherein the compound isadministered transdermally.
 54. The method of claim 50 wherein thecompound is administered rectally.
 55. The method of claim 50 whereinthe compound is administered nasally.
 56. The method of claim 50 whereinthe compound is administered sublingually.
 57. The method of claim 50wherein the compound is administered in a dosage of from about 0.01μg/day to about 1000 μg/day.